Characterizing Lignocellulose Breakdown Mechanisms in Anaerobic Gut Fungi
Shiyan Jin1* (ShiyanJin@lbl.gov), Stephen P. Lillington1, Kai Deng2, Thomas S. Lankiewicz1, Trent R. Northen2,3, Christopher J. Petzold2,3, Steven Singer2,3, Blake A. Simmons2,3, Michelle A. O’Malley1, and Jay Keasling2,3
1University of California–Santa Barbara; 2Joint BioEnergy Institute; and 3Lawrence Berkeley National Laboratory
The vision of Joint Bioenergy Institute (JBEI) is that bioenergy crops can be converted into economically viable, carbon-neutral, biofuels and renewable chemicals currently derived from petroleum, and many other bioproducts that cannot be efficiently produced from petroleum.
Lignocellulose is an attractive feedstock for renewable chemical manufacturing and bio-based chemical production, which reduces dependency on petroleum, but its recalcitrant structure requires multiple catalytic steps for sufficient hydrolysis, rendering current production technologies energetically and economically expensive. Anaerobic gut fungi (AGF) provide an attractive alternative strategy for biomass valorization by secreting a variety of carbohydrate-active enzymes (CAZymes) to efficiently degrade unpretreated biomass (Solomon et al. 2016). Many CAZymes are colocalized in fungal cellulosomes (enzyme complexes) that are thought to accelerate lignocellulose breakdown compared to the action of freely diffusing enzymes (Lillington et al. 2021). Anaerobic gut fungi control cellulosome compositions by regulating the production of biomass-degrading enzymes based on fungal life stage and the complexity of available substrates, as supported by advanced microscopy imaging and transcriptomic characterization (Solomon et al. 2016; Lillington et al. 2021). To further assess the differences in mechanistic properties among cellulosomes, researchers first developed an isolation technique to harvest these complexes from fungi grown on aqueous cellulose (cellobiose), fibrous cellulose (Whatman filter paper) and lignocellulose (reed canary grass), followed by protein purification via fast protein liquid chromatography. Researchers used nanostructure-initiator mass spectrometry (NIMS) probes, which quantify oligosaccharide abundance in solution, to measure cellulases and hemicellulase activity based on the hydrolysis of NIMS probes (Deng et al. 2014). Amorphous cellulose probes, crystalline cellulose probes and hemicellulose probes were used to detect any preference in degradation among enzyme complexes. Researchers observed that cellulosomes produced when growing fungi on lignocellulose were the most active and digested all three NIMS probes at a comparable level. Additionally, cellulosomes secreted when grown on filter paper, even though they perform better in cellulose digestion than that grown on cellobiose, exhibited a strong preference toward cellulose over hemicellulose. Currently, researchers are screening a library of 200+ synthesized genes from fungal cellulosomes to unmask their functions and roles in lignocellulose degradation via a combination of proteomics and NIMS characterization.
Additionally, there is evidence from two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC-NMR) spectrometry, which suggests an up to 8% reduction in β-aryl ether linkage, a typical lignin bond, in addition to the changes in lignin monomer composition after incubating biomass (sorghum, switchgrass and poplar) with Neocallimastix californiae and Anaeromyces robustus, two AGF strains. The same phenomenon is observed in lignin content analysis and gel permeable chromatography. This is the first time that lignin modification is observed with high confidence under an anaerobic environment. Researchers are working on expanding the biomass library to include different types of softwood and hardwood, to develop an increased mechanistic understanding of anaerobic lignin modification in plants with different lignin compositions. Moreover, researchers are developing and applying lignin-based probes to characterize possible mechanisms of anaerobic lignin breakdown.
Solomon, K. V., et al. 2016. “Early-Branching Gut Fungi Possess a Large, Comprehensive Array of Biomass-Degrading Enzymes.” Science 351(6278), 1192–95.
Lillington, S. P., et al. 2021. “Cellulosome Localization Patterns Vary Across Life Stages of Anaerobic Fungi.” mBio 12(3), e00832-00821.
Deng, K., et al. 2014. “Rapid Kinetic Characterization of Glycosyl Hydrolases Based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)”. ACS Chemical Biology 9(7), 1470–79.
This work was part of the DOE Joint BioEnergy Institute (http:// www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.